The immune system is a highly complex network of interacting cells, receptors and factor that combine to yield a potent immune response. Over the last decade, major advances in the study of the immune system has depended on our ability to examine individual cells of the immune network. Automated flow cytometry using fluorescence activated cell sorters (FACS) has enabled immunologists to analyze cell surface as well as intracellular proteins and DNA on as few as 1% of the cells. More recently, FACS has become an even more powerful tool capable of examining rapid intracellular events such as apoptosis, cell cycle events and cell signalling (Ca++flux) in individual cells of multicell conjugates. Equally important is the preparative sorting capability of these machines. These instruments an separate, with extremely high efficiency, individual subpopulations of cells and yield tens of millions of cells with a purity of greater than 99%. Access to these instruments is critical to the Program Project members. Every project depends on both analytical and preparative flow cytometry. Drs. Herold and Bluestone will use the facility to analyze the cell surface phenotype of APCs, activated T cells and islets from normal NOD and tolerant mice. They will prepare purified T cells from NOD and TCR transgenic mice for adoptive transfer studies. Dr Sant will use flow cytometry to analyze class II transfectants, newly-derived transgenic mice and natural APC populations. Dr. Miller will depend on flow cytometry to analyze TCR transgenic mice and select for new monoclonal antibodies specific for novel co-stimulatory molecules. Dr. Thompson has developed a highly sensitive method for examine apoptosis using flow cytometry to analyze individual cells. The cell sorter facility is, therefore, an essential resource for the research proposed in the program project.